This invention relates to arrays of biopolymers used in diagnostics, screening, gene expression analysis, and other applications. In particular, the invention relates to a method of fabricating biopolymer arrays, such as DNA arrays.
Polynucleotide arrays, such as DNA or RNA arrays, are known and are used, for example, as diagnostic or screening tools. Such arrays comprise a plurality of different polynucleotide probes arranged in a predetermined configuration on a substrate. The polynucleotides of the plurality differ by having a different nucleotide sequence. Different polynucleotide probes are located at different regions (also known as features or spots) on the substrate, wherein in each region, multiple copies of the same polynucleotide are usually present.
The array is exposed to a sample of biological material to be evaluated, also known as the xe2x80x9ctargetxe2x80x9d. Upon exposure to the target sample, the array will exhibit a binding pattern, wherein complementary target polynucleotides will hybridize or bind to the array polynucleotide probes during an assay. This binding pattern can be observed, for example, by labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescence pattern on the array. Assuming that the different sequence polynucleotide probes were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the target sample.
Biopolymer arrays can be fabricated using either methods of deposition of intact biopolymer species or using in situ synthesis methods. The deposition methods basically involve depositing intact biopolymers at predetermined locations on a substrate that are suitably activated such that the intact biopolymers can link thereto. The intact species of biopolymers, each having different monomer sequences, may be deposited at different regions of the substrate to yield the completed array having a predetermined configuration. Typical procedures known in the art for deposition of intact polynucleotide species, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the substrate surface, some of the fluid is transferred from the pin or capillary to the substrate location. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for the plurality of different polynucleotides and, eventually, the desired array having a predetermined configuration is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere.
The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA) using phosphoramidite or other chemistry. Such in situ synthesis methods can be basically regarded as iterative steps of depositing droplets of. (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one cycle so that the different regions of the completed array will carry the plurality of different biopolymer sequences as desired in the completed array. In situ synthesis methods may require one or more intermediate further steps in each iteration, such as oxidation and washing steps, as are well known in the art.
In order for an assay to yield accurate results, it is important that the different biopolymer features actually be present on the array, that they are put down accurately in the desired or predetermined pattern, that the biopolymers are of the correct size, and that each different feature be uniformly populated with the respective biopolymer.
In polynucleotide arrays, the conventional in situ synthesis methods use phosphoramidite nucleoside monomers. In order for the phosphoramidite group to link to a hydroxyl of a previously deposited deprotected polynucleotide monomer, it must first be activated usually by using a weak acid, such as tetrazole. However, an activated phosphoramidite is highly reactive with moisture in the air. Therefore, unless some precaution is taken, the activated phosphoramidite can be used up before the desired reaction is complete. As a result there is a reduction in the deposited phosphoramidite monomer available for forming the complete polynucleotide. This problem is present even when the synthesis is performed in a nitrogen chamber.
Further, the size (volume) of the synthesis droplet on the substrate surface could be very small, such as a few pico- or nano-liters, such that the ratio of surface to volume is very high. A high surface to volume ratio favors the diffusion of moisture into the droplets. Initially, the moisture from the air tends to be adsorbed at the surface of the synthesis droplet. Therefore, the phosphoramidite concentration at the surface of the droplet will tend to be lowest. Consequently, the concentration of a completed probe polynucleotide at a feature on the array tends to decrease from the center of a feature toward its perimeter. Variations in completed probe concentration within a feature result in a decrease in the concentration of target sample that consequently hybridizes to the respective polynucleotide probe. Therefore, the total signal that should be available from the hybridized target is diminished at the particular feature location during optical evaluation of the array. Further, it should be noted that the water vapor concentration in the ambient atmosphere might vary. Therefore, the signal from the hybridized target may also vary from array to array, leading to inconsistency in absolute signal generated from different arrays of a batch when the same concentration of a target is encountered.
The foregoing problems exist particularly where the phosphoramidite is mixed with the activator and the mixture is deposited as a droplet on the substrate, and even where the activator is deposited onto a previously deposited droplet containing the phosphoramidite, both as such are described in PCT publication WO 98-41531. In either case, ambient moisture presents a problem. Furthermore, when one droplet is deposited on the other, there is no guarantee of efficient mixing such that the activated phosphoramidite will be evenly present at the substrate surface.
Thus, it would be advantageous to have a means of fabricating biopolymer arrays that lessens the likelihood of deleterious environmental influences on the accuracy of the fabrication. In particular, it would be desirable, in the fabrication of arrays of biopolymers using biomonomers with a linking group that must be activated (such as a phosphoramidite), to provide a means by which the potential reactivity of the activated biomonomer with an ambient atmosphere component (such as water vapor in air) can be kept low.
The present invention provides a method of fabricating an array of biopolymers on a substrate. The method is useful for shielding biosynthesis reactions and the bio-reactants from the ambient environment. Moreover, the method is useful for shielding pre-synthesized biopolymers during their attachment to an array substrate. In particular, the method is useful for shielding biosynthesis reactions and reaction components during the synthesis process or linking process that are susceptible to reaction with a component of the ambient environment, for example moisture in the air. The method of the invention is applicable to the conventional fabrication and synthesis methods used to fabricate a biopolymer array.
In one aspect of the method, the array is fabricated using conventional in situ techniques. A first biomonomer is deposited onto a substrate for linking to a surface of the substrate in an array pattern of features by conventional methods. The linked biomonomer is deprotected using conventional methods, such that the biomonomer can react with subsequent biomonomers that are added to grow the biopolymer chain. The subsequent biomonomer may require activation before attachment to the growing chain. The biomonomer and a suitable activation reagent are deposited on the array for attachment to the deprotected surface-linked biomonomer. The biomonomers are deposited using conventional deposition equipment, such as computer controlled inkjet systems or piezoelectric deposition systems that are well known in the art. The method of the invention comprises applying a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized. The NMF is inert and insoluble with the ancillary materials, reagents and biomonomers used in the synthesis of the biopolymers. In accordance with the invention, the NMF provides a shield between the ambient atmosphere and the biopolymer synthesis materials at the surface of the array during the synthesis process. The shield will delay the diffusion of ambient conditions into the synthesis areas. The subsequent biomonomers are deposited on the array until a desired biopolymer sequence is synthesized at each feature. The NMF may be applied as droplets over each feature location or may be applied by flooding the surface of the array to fully cover the features and the growing biopolymer sequences.
The deposition system used to deposit biomonomers in solution on the array has a head with multiple pulsejets, each of which can dispense fluid droplets onto the substrate. Each such jet includes a chamber with an orifice, and an ejector which, when activated, causes a droplet to be ejected from the orifice. According to the invention, the inkjets of the deposition system are either immersed into the NMF to fire droplets of the activated biomonomer through the NMF to the biopolymer synthesis sites, or not immersed, but positioned above the level of the NMF to fire the activated biomonomer droplets into the NMF to the biopolymer synthesis sites on the array. In the preferred embodiment, the density of the NMF is different from the anhydrous solution of solvent, biomonomers and activation reagents.
In a preferred embodiment, the method of fabricating further comprise the step of deactivating any unreacted activation reagent after the activated biomonomer is added to the linked biomonomer on the array. An ancillary material that stops the action of the activation reagent (i.e., deactivation reagent) may be added to the array, preferably by flooding the array surface. The deactivation reagent solution has a density that is different from the density of the NMF to facilitate the deactivation reagent reaching the unreacted activation reagent at biopolymer synthesis sites through the NMF shield. The preferred embodiment further comprises the step of removing all the ancillary materials and unreacted biomonomer from the array surface so that the growing biopolymer chain can undergo other chemistry.
The foregoing steps are repeated, with a biomonomer deposited and linked to a previously deposited and linked biomonomer on the substrate. The growing biopolymer chain acts as a substrate bound moiety for each cycle, until all of the biomonomers have been added to the biopolymer array. In the fabrication of a typical array with multiple features, all of the foregoing steps are repeated at each of multiple different regions on the same substrate, where it is desired to form the biopolymer features.
The biopolymers arrays that may be fabricated according to the invention include DNA, RNA, proteins, etc. arrays, for example. Where the array is a polynucleotide or oligonucleotide array (for example, DNA), the biomonomer is a nucleoside monomeric unit. The activated biomonomer is typically a phosphoramidite according to conventional oligonucleotide synthesis. Activated phosphoramidites are well known to be highly reactive with moisture. Without the method of the invention, an activated phosphoramidite will react with water vapor in ambient atmosphere and be depleted before a sufficient amount of the phosphoramidite has reacted with the growing polynucleotide chains of the array, even in a nitrogen chamber.
In another aspect of the invention, a method of fabricating biopolymer arrays from pre-synthesized biopolymers is provided. The pre-synthesized biopolymer is deprotected before it is linked to the array surface. The deprotected pre-synthesized biopolymer is soluble in aqueous buffer solution. The droplets of the deprotected pre- synthesized biopolymer solution that are deposited for linking to an array substrate are very small and have the tendency to evaporate quickly in the ambient environment. The method of fabricating according to this embodiment comprises enclosing the droplets of the deprotected pre-synthesized biopolymer solution in the NMF for deposition. The NMF is inert, immiscible and insoluble in aqueous solution. Therefore, the NMF will surround the droplets and delay the diffusion of the aqueous solution out of the droplet such that the concentration of the deprotected pre-synthesized biopolymer will remain relatively constant while it links to the surface of the array substrate at each feature.
In still another aspect of the invention, a method of shielding biosynthesis reactions and biosynthesis reactants from the ambient environment is provided. The method of shielding comprises applying the NMF to one or more sites where the biosynthesis reactions take place. The NMF is inert and insoluble with respect to the biosynthesis reactions and the biosynthesis reactants. The NMF is applied to cover the biosynthesis site(s). The method of shielding further comprises depositing one or more of the sensitive biosynthesis reactants through the NMF on the biosynthesis site(s).
In still another aspect of the invention, a shield that protects sensitive biosynthesis reactions and biosynthesis reactants from the ambient environment is provided. The shield comprises a non-miscible fluid (NMF) applied to cover the biosynthesis reactions and reactants. The NMF is inert and insoluble with respect to the biosynthesis reactions and the biosynthesis reactants.
The present methods and apparatus provide any one or more of a number of useful benefits. For example, in the fabrication of arrays of biopolymers using biomonomers with a linking group that must be activated, the present invention provides a means by which the potential reactivity of the activated biomonomer with an ambient atmosphere component can be kept low. Further, in the fabrication of arrays of biopolymers using pre-synthesized biopolymers that are water-soluble when deprotected for linking to the surface of the substrate, the present invention provides a means by which the potential reactivity of the deprotected biopolymer in solution with an ambient atmosphere can be kept low.